Fibrin, formed in response to vascular injury, constitutes the protein polymeric scaffold known as the provisional extracellular matrix. Fibrin serves as the temporary initial scaffolding for the invasion of inflammatory and repair cells into wound environments. Fibrinogen, the inactive precursor to fibrin, is a 340 kDa plasma glycoprotein that circulates in blood plasma at concentrations of 2 to 4 mg/mL. As a part of its normal functions, fibrin(ogen) features a variety of binding sites for self-regulatory enzymes (including thrombin, factor XIII, plasminogen, tissue plasminogen activator), ECM components, growth factors, cytokines and cell receptors; components that are critical in the remodeling of the matrix as part of the wound healing response (Laurens N, et al. J Thromb Haemost 2006, 4(5):932-939; Mosesson M W. J Thromb Haemost 2005, 3(8):1894-1904). Due to the ease of purification of this highly expressed plasma protein and its native role in the guidance of wound repair, the fibrinogen/fibrin system is employed in a number of surgical hemostats and tissue sealants (FDA-approved products include Tisseel®, Evicel™) and is actively used as a biomaterial for developing therapeutic strategies in regenerative medicine (Spotnitz W D, et al. Transfusion 2008; 48(7):1502-1516; Ahmed T A, et al. Tissue Eng Part B Rev 2008, 14(2):199-215; Breen A, et al. Tissue Eng Part B Rev 2009, 15(2): 201-214).
Significant efforts have been made to develop and enhance fibrin as a tissue scaffold and drug delivery vehicle through both modification of the molecule and engagement of the native polymer system biology. For instance, functionalized polyethylene glycol was explored as a means to covalently tether therapeutic proteins to fibrinogen's backbone thus enabling their delivery in fibrin polymers (Barker T H, et al. J Biomed Mater Res 2001, 56(4):529-535). Alternatively, factor XIIIa, also responsible for the covalent attachment of other non-fibrin proteins (e.g. fibronectin, α2-plasmin inhibitor) to the C-terminus of the Aα chains in vivo, has been shown to be a reproducible method of crosslinking RGD-containing peptides into fibrin gels during coagulation (Schense J C, et al. Bioconjug Chem 1999, 10(1):75-81). The factor XIIIa substrate sequence has subsequently been used for the incorporation of recombinantly-produced growth factors, cell adhesion molecules and morphogens into fibrin matrices, usually with an additional protease-sensitive cleavage site to facilitate their release from fibrin (Ehrbar M, et al. J Control Release 2005, 101(1-3):93-109; Schmoekel H G, et al. Biotechnol Bioeng 2005, 89(3):253-262; Pittier R, et al. J Neurobiol 2005, 63(1):1-14; Arrighi I, et al. Biomaterials 2009, 30(9):1763-0.1771; Hall H, et al. Microvasc Res 2004, 68(3):169-178). As an intriguing spin-off, bi-domain peptides comprising the factor XIIIa substrate sequence and the heparin-binding domain have been used to increase the concentration of fibrin-bound heparin, thereby improving the retention characteristics of heparin-binding growth factors (Willerth S M, et al. J Biomed Mater Res A 2007, 80(1):13-23; Wood M D, et al. J Biomed Mater Res A 2008, 84(2):300-312; Sakiyama-Elbert S E, et al. J Control Release 2000, 65(3):389-402; Sakiyama-Elbert S E, et al. J Control Release 2000, 69(1):149-158). In particular, such biomimetic affinity-based systems are advantageous for the local delivery of factors that are internalized by cells as part of the signaling process. These methods, however, require intermediary molecules or enzymes, like factor XIIIa, to conjugate the agents to fibrin, and the desired deliverable, such as a drug or protein, can not be incorporated prior to the initiation of polymer.
It is an object of the invention to provide improved methods of incorporating therapeutic agents into a fibrin polymer.
It is a further object of the invention to target substances to fibrinogen and fibrin within a subject.
It is a further object of the invention to provide methods for detecting fibrin polymers in a subject.
It is a further object of the invention to promote wound healing in a subject.
It is a further object of the invention to provide fibrin polymers with modified physical properties.